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Research Article


Development of a multiplex polymerase chain reaction assay for the detection of Piroplasma (Babesia spp. and Theileria spp.), Anaplasma spp., and Trypanosoma evansi in cattle

Jiravich Methawiroon, Piangjai Chalermwong, Ornarin Boonjint, Piangdow Chiawvitkan, Sakulchit Wichianchot, Chanapath Thabthimsri, Preeyanuch Thongpoo, Ketsarin Kamyingkird, Sathaporn Jittapalapong, Eukote Suwan.


Abstract
Background:
Blood parasites cause serious drawbacks in the livestock industry. The detection methods rely on microscopic examination and polymerase chain reaction (PCR), which requires an expert person, labor, time, and cost.

Aim:
In this study, multiplex polymerase chain reaction (PCR) has been developed for hemoparasite consisting of Piroplasma (Babesia spp. and Theileria spp.), Anaplasma spp., and Trypanosoma evansi detections.

Methods:
Blood parasite infections in cattle were investigated using 3 different methods: microscopic examination, single PCR, and multiplex PCR. The Anaplasma 16s rRNA gene, Babesia 18s rRNA gene, Theileria MPSP gene, and Trypanosome ITS1 gene have been used to detect Anaplasma spp., Babesia spp., Theileria spp., and T. evansi, respectively, using molecular methods. The sensitivity and specificity of multiplex PCR were evaluated. Multiplex PCR results were compared with microscopic examination and single PCR.

Results:
Multiplex PCR assay revealed a limit of detection of 0.01–10 pg of parasite DNA. According to the evaluation of 60 bovine blood samples, blood smear, single PCR, and multiplex PCR revealed 50.0%, 18.3%, and 26.7% of single infections, and 40.0%, 81.7%, and 50.0% of co-infections, respectively. The comparative analysis between multiplex PCR with microscopic examination and single PCR revealed that triple infection, Babesia spp., Theileria spp., and T. evansi, showed 50.0% sensitivity, 100% specificity and PPV, 98% NPV, and substantial agreement indicated by a Cohen’s Kappa value of 0.659.

Conclusion:
The multiplex PCR assay developed in this study may be helpful for improved hemoparasite prevention and control when combined with farmer education, proper hygiene practices, and effective environmental management.

Key words: Hemoparasites; Multiplex PCR; Piroplasma; Trypanosoma evansi.


 
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How to Cite this Article
Pubmed Style

Methawiroon J, Chalermwong P, Boonjint O, Chiawvitkan P, Wichianchot S, Thabthimsri C, Thongpoo P, Kamyingkird K, Jittapalapong S, Suwan E. Development of a multiplex polymerase chain reaction assay for the detection of Piroplasma (Babesia spp. and Theileria spp.), Anaplasma spp., and Trypanosoma evansi in cattle. Open Vet. J.. 2026; 16(5): 3218-3228. doi:10.5455/OVJ.2026.v16.i5.61

Web Style

Methawiroon J, Chalermwong P, Boonjint O, Chiawvitkan P, Wichianchot S, Thabthimsri C, Thongpoo P, Kamyingkird K, Jittapalapong S, Suwan E. Development of a multiplex polymerase chain reaction assay for the detection of Piroplasma (Babesia spp. and Theileria spp.), Anaplasma spp., and Trypanosoma evansi in cattle. https://www.openveterinaryjournal.com/?mno=289481 [Access: June 01, 2026]. doi:10.5455/OVJ.2026.v16.i5.61

AMA (American Medical Association) Style

Methawiroon J, Chalermwong P, Boonjint O, Chiawvitkan P, Wichianchot S, Thabthimsri C, Thongpoo P, Kamyingkird K, Jittapalapong S, Suwan E. Development of a multiplex polymerase chain reaction assay for the detection of Piroplasma (Babesia spp. and Theileria spp.), Anaplasma spp., and Trypanosoma evansi in cattle. Open Vet. J.. 2026; 16(5): 3218-3228. doi:10.5455/OVJ.2026.v16.i5.61


Vancouver/ICMJE Style

Methawiroon J, Chalermwong P, Boonjint O, Chiawvitkan P, Wichianchot S, Thabthimsri C, Thongpoo P, Kamyingkird K, Jittapalapong S, Suwan E. Development of a multiplex polymerase chain reaction assay for the detection of Piroplasma (Babesia spp. and Theileria spp.), Anaplasma spp., and Trypanosoma evansi in cattle. Open Vet. J.. (2026), [cited June 01, 2026]; 16(5): 3218-3228. doi:10.5455/OVJ.2026.v16.i5.61


Harvard Style

Methawiroon, J., Chalermwong, . P., Boonjint, . O., Chiawvitkan, . P., Wichianchot, . S., Thabthimsri, . C., Thongpoo, . P., Kamyingkird, . K., Jittapalapong, . S. & Suwan, . E. (2026) Development of a multiplex polymerase chain reaction assay for the detection of Piroplasma (Babesia spp. and Theileria spp.), Anaplasma spp., and Trypanosoma evansi in cattle. Open Vet. J., 16 (5), 3218-3228. doi:10.5455/OVJ.2026.v16.i5.61


Turabian Style

Methawiroon, Jiravich, Piangjai Chalermwong, Ornarin Boonjint, Piangdow Chiawvitkan, Sakulchit Wichianchot, Chanapath Thabthimsri, Preeyanuch Thongpoo, Ketsarin Kamyingkird, Sathaporn Jittapalapong, and Eukote Suwan. 2026. Development of a multiplex polymerase chain reaction assay for the detection of Piroplasma (Babesia spp. and Theileria spp.), Anaplasma spp., and Trypanosoma evansi in cattle. Open Veterinary Journal, 16 (5), 3218-3228. doi:10.5455/OVJ.2026.v16.i5.61


Chicago Style

Methawiroon, Jiravich, Piangjai Chalermwong, Ornarin Boonjint, Piangdow Chiawvitkan, Sakulchit Wichianchot, Chanapath Thabthimsri, Preeyanuch Thongpoo, Ketsarin Kamyingkird, Sathaporn Jittapalapong, and Eukote Suwan. "Development of a multiplex polymerase chain reaction assay for the detection of Piroplasma (Babesia spp. and Theileria spp.), Anaplasma spp., and Trypanosoma evansi in cattle." Open Veterinary Journal 16 (2026), 3218-3228. doi:10.5455/OVJ.2026.v16.i5.61


MLA (The Modern Language Association) Style

Methawiroon, Jiravich, Piangjai Chalermwong, Ornarin Boonjint, Piangdow Chiawvitkan, Sakulchit Wichianchot, Chanapath Thabthimsri, Preeyanuch Thongpoo, Ketsarin Kamyingkird, Sathaporn Jittapalapong, and Eukote Suwan. "Development of a multiplex polymerase chain reaction assay for the detection of Piroplasma (Babesia spp. and Theileria spp.), Anaplasma spp., and Trypanosoma evansi in cattle." Open Veterinary Journal 16.5 (2026), 3218-3228. Print. doi:10.5455/OVJ.2026.v16.i5.61


APA (American Psychological Association) Style

Methawiroon, J., Chalermwong, . P., Boonjint, . O., Chiawvitkan, . P., Wichianchot, . S., Thabthimsri, . C., Thongpoo, . P., Kamyingkird, . K., Jittapalapong, . S. & Suwan, . E. (2026) Development of a multiplex polymerase chain reaction assay for the detection of Piroplasma (Babesia spp. and Theileria spp.), Anaplasma spp., and Trypanosoma evansi in cattle. Open Veterinary Journal, 16 (5), 3218-3228. doi:10.5455/OVJ.2026.v16.i5.61


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